Abstract
To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that cyp26a1 in zebrafish and its paralog Cyp26b1 in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish.
Highlights
A critical stage in vertebrate sex determination is entry of the bipotential gonadal primordium into a developmental pathway leading to ovary or testis
To critically test the hypothesis that reciprocal gene loss occurred in the Cyp26 family in the zebrafish and mouse lineages leading to erroneous orthology assignments, we examined a data set independent of Cyp26 amino acid sequence by examining syntenic conservation within the genomic neighborhoods (GN) surrounding Cyp26 genes in zebrafish and mouse (Figure 2B–F) and medaka (Figure S1, Figure S2)
Our findings reveal several significant differences between retinoic acid (RA)-regulated gonadogenesis in zebrafish and tetrapods, including which cells express RAsynthesizing enzymes, which paralog encodes gonadal RAdegrading enzymes, whether RA-degrading enzymes are expressed in a dimorphic fashion, whether Stra8 regulates entry into meiosis, and whether the onset of meiosis is sexually dimorphic
Summary
A critical stage in vertebrate sex determination is entry of the bipotential gonadal primordium into a developmental pathway leading to ovary or testis (reviewed in 1). Discovery of Sry (sex-determining region Y), the major sex-determining gene in mammals [2,3], stimulated the search for genetic mechanisms that control the sexual fate of somatic and germ cells during vertebrate gonadogenesis. In female embryonic mouse gonads, germ cells enter into meiosis at 13.5 days post-coitum (dpc) and concomitantly, the somatic gonadal primordium initiates ovarian differentiation by developing into granulosa and theca cells. In male embryonic mouse gonads, SRY-expressing pre-Sertoli cells initiate testicular differentiation and germ cells arrest in the G0/G1 phase of the mitotic cell cycle, postponing meiosis until after birth [20,21]. Meiotic XX germ cells antagonize testicular development in XY gonads in tissue co-culture experiments, leading to the hypothesis that germ cells committed to meiosis reinforce ovarian fate by antagonizing the testis pathway [22]
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